Quinone reductase (QR1), a cytosolic FAD-containing flavoprotein, is induced as part of the chemoprotective phase II enzymes by many xenobiotics. It is widely distributed in mammalian tissues where it plays a major role in the detoxication of quinones and their metabolic precursors by promoting the obligatory two-electron reductions of many quinones to hydroquinones. In addition to its role in the detoxification of dietary quinones, the enzyme has been shown to catalyze the reductive activation of quinolic cancer chemotherapeutic compounds. Since QR1 expression is enhanced expression in certain tumors, particularly lung carcinoma, colon, liver, and breast tumors, and non-small cell lung cancer, it represents an ideal target for the development of cancer chemotherapeutic compounds. As part of this project we carried out structural and biochemical studies that provided the first major insight into the mechanism of the enzyme: we determined the structures of several QRs and proposed the mechanism that is widely accepted in the field. In addition, we analyzed the complexes of several cancer chemotherapeutic prodrugs with QR1 and inferred tentative rules that can be used to design new compounds with improved characteristics. This project deals with the two aspects of QR1 physiology: chemotherapy and chemoprotection. Our studies on QR biochemistry are focusing on the activation of chemotherapeutic compounds as understanding the mechanism and the specificity of the enzyme has a direct impact on the development of more effective compounds. We propose to continue our studies to elucidate the mechanism of QR1, to characterize the specificity of QR1 for chemotherapeutic agents, and to complete the elucidation of the function and the mechanism of QR2. Chemoprotection by QR is part of the coordinated response that involves the induction of phase II enzymes. As induction of chemoprotection enzymes is the stage of this important process most amenable for therapeutic interventions, our studies in this area will focus on the mechanism of induction. One protein, Keap1, is thought to be the chemical sensor that initiates the response of oxidative stress. We propose to study this protein as well as its complexes with its partner protein Nrf2 to explore the primary chemical mechanism by which inducers trigger the release of Nrf2 from inactivation by Keap1.